Magneto-Optical Relaxation Measurements of Functionalized Nanoparticles as a Novel Biosensor

Measurements of magneto-optical relaxation signals of magnetic nanoparticles functionalized with biomolecules are a novel biosensing tool. Upon transmission of a laser beam through a nanoparticle suspension in a pulsed magnetic field, the properties of the laser beam change. This can be detected by optical methods. Biomolecular binding events leading to aggregation of nanoparticles are ascertainable by calculating the relaxation time and from this, the hydrodynamic diameters of the involved particles from the optical signal. Interaction between insulin-like growth factor 1 (IGF-1) and its antibody was utilized for demonstration of the measurement setup applicability as an immunoassay. Furthermore, a formerly developed kinetic model was utilized in order to determine kinetic parameters of the interaction. Beside utilization of the method as an immunoassay it can be applied for the characterization of diverse magnetic nanoparticles regarding their size and size distribution

Effect of Methylene Blue Pathogen Inactivation on the Integrity of Immunoglobulin M and G

Introduction: In the light of the ongoing SARS-CoV-2 pandemic, convalescent plasma is a treatment option for CO­VID-19. In contrast to usual therapeutic plasma, the therapeutic agents of convalescent plasma do not represent clotting factor activities, but immunoglobulins. Quarantine storage of convalescent plasma as a measure to reduce the risk of pathogen transmission is not feasible. Therefore, pathogen inactivation (e.g., Theraflex®-MB, Macopharma, Mouvaux, France) is an attractive option. Data on the impact of pathogen inactivation by methylene blue (MB) treatment on antibody integrity are sparse. Methods: Antigen-specific binding capacity was tested before and after MB treatment of plasma (n = 10). IgG and IgM isoagglutinin titers were tested by agglutination in increasing dilutions. Furthermore, the binding of anti-EBV and anti-tetanus toxin IgG to their specific antigens was assessed by ELISA, and IgG binding to Fc receptors was assessed by flow cytometry using THP-1 cells expressing FcRI and FcRII. Results: There was no significant difference in the isoagglutinin titers, the antigen binding capacity of anti-EBV and anti-tetanus toxin IgG, as well as the Fc receptor binding capacity before and after MB treatment of plasma. Conclusion: MB treatment of plasma does not inhibit the binding capacity of IgM and IgG to their epitopes, or the Fc receptor interaction of IgG. Based on these results, MB treatment of convalescent plasma is appropriate to reduce the risk of pathogen transmission if quarantine storage is omitted

Divalent magnesium restores cytoskeletal storage lesions in cold-stored platelet concentrates

Cold storage of platelet concentrates (PC) has become attractive due to the reduced risk of bacterial proliferation, but in vivo circulation time of cold-stored platelets is reduced. Ca2+ release from storage organelles and higher activity of Ca2+ pumps at temperatures < 15 °C triggers cytoskeleton changes. This is suppressed by Mg2+ addition, avoiding a shift in Ca2+ hemostasis and cytoskeletal alterations. We report on the impact of 2–10 mM Mg2+ on cytoskeleton alterations of platelets from PC stored at room temperature (RT) or 4 °C in additive solution (PAS), 30% plasma. Deformation of platelets was assessed by real-time deformability cytometry (RT-DC), a method for biomechanical cell characterization. Deformation was strongly affected by storage at 4 °C and preserved by Mg2+ addition ≥ 4 mM Mg2+ (mean ± SD of median deformation 4 °C vs. 4 °C + 10 mM Mg2+ 0.073 ± 0.021 vs. 0.118 ± 0.023, p < 0.01; n = 6, day 7). These results were confirmed by immunofluorescence microscopy, showing that Mg2+  ≥ 4 mM prevents 4 °C storage induced cytoskeletal structure lesion. Standard in vitro platelet function tests showed minor differences between RT and cold-stored platelets. Hypotonic shock response was not significantly different between RT stored (56.38 ± 29.36%) and cold-stored platelets with (55.22 ± 11.16%) or without magnesium (45.65 ± 11.59%; p = 0.042, all n = 6, day 1). CD62P expression and platelet aggregation response were similar between RT and 4 °C stored platelets, with minor changes in the presence of higher Mg2+ concentrations. In conclusion, increasing Mg2+ up to 10 mM in PAS counteracts 4 °C storage lesions in platelets, maintains platelet cytoskeletal integrity and biomechanical properties comparable to RT stored platelets

Magnetic Nanoparticle Labeling of Human Platelets from Platelet Concentrates for Recovery and Survival Studies

Platelets are the smallest blood cells and important for hemostasis. Platelet concentrates (PC) are medicinal products transfused to prevent or treat bleeding. Typically, platelets in PCs are assessed by in vitro tests for their function. However, in vivo testing of these platelets is highly desirable. To distinguish transfused platelets from patients or probands own cells after PC transfusions within the scope of clinical studies, platelets need to be efficiently labeled with minimal preactivation prior to transfusion. Here we report on a method for improved cell uptake of ferucarbotran magnetic nanoparticles contained in Resovist, an FDA-approved MRI contrast agent, by modifying the nanoparticle shell with human serum albumin (HSA). Both HSA-ferucarbotran nanoparticles and magnetically labeled platelets were produced according to EU-GMP guidelines. Platelet function after labeling was evaluated by light transmission aggregometry and by determination of expression of CD62P as platelet activation marker. Magnetic labeling does not impair platelet function and platelets showed reasonable activation response to agonists. Platelet survival studies in NOD/SCID-mice resulted in comparable survival behavior of magnetically labeled and nonlabeled platelets. Additionally, labeled platelets can be recovered from whole blood by magnetic separation

GPVI expression is linked to platelet size, age, and reactivity

Platelets within one individual display heterogeneity in reactivity, size, age, and expression of surface receptors. To investigate the combined intra-individual contribution of platelet size, platelet age, and receptor expression levels on the reactivity of platelets, we studied fractions of large and small platelets from healthy donors separated by differential centrifugation. Size-separated platelet fractions were perfused over a collagen-coated surface to assess thrombus formation. Multicolour flow cytometry was used to characterise resting and stimulated platelet subpopulations. Platelet age was determined based on RNA and HLA-I labelling. Signal transduction was analysed by measuring consecutive phosphorylation of serine/threonine-protein kinase Akt. Large platelets adhered faster to collagen under flow and formed larger thrombi, compared to small platelets. Among the large platelets a highly reactive juvenile platelet subpopulation was identified with high GPVI expression. Elevated GPVI expression correlated with high HLA-I expression, RNA content and increased platelet reactivity. Akt phosphorylation and activation upon collagen stimulation differed stronger between juvenile and older platelets than between large and small platelets. GPVI expression and platelet reactivity decreased throughout platelet storage at 22°C and was better maintained throughout cold storage at 4°C. We further detected a higher GPVI expression in platelets of patients with immune thrombocytopenia. Our findings show that high GPVI expression is a feature of highly reactive juvenile platelets, which are predominantly found among the large platelet population explaining the better performance of large platelets during thrombus formation. These data are important for studies of thrombus formation, platelet storage and ITP